Rabbit anti ift20

Cells in microslides were washed twice with ice-cold PBS and lysed in ice with 150 µl of 1X Laemmli buffer (60 mM Tris-HCL pH = 6.8, 2% SDS, 10% Glycerol, bromophenol blue, supplemented with 100 mM DTT) for 30 min. Samples were boiled for 10 min at 95 °C, separated by SDS/PAGE and then transferred onto Nitrocellulose membranes. Western blot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore). The following antibodies were used in immunoblotting: rabbit-anti LC3 (1:1000, Sigma, Cat#L7543); mouse-anti-actin (1:5000, Millipore, Cat#1501); mouse-anti-Beclin1 (1:2000, BD Biosciences,Cat#612113); rabbit-anti-IFT88 (1:1000, Proteintech, Cat#13967-1-AP); rabbit-anti-PIK3C2α (1:800, Novus, Cat#NBP2-19829); mouse-anti-Wipi2 (2A2) (1:1000, AbD Serotec, Cat#MCA5780GA); rabbit-anti-Rab11 (1:1000, Cell Signaling, Cat#2413S); rabbit-anti-VPS34 (d9A5) (1:500, Cell Signaling, Cat#4263); rabbit-anti-LKB1 (1:1000, Cell signaling, Cat#3050); rabbit-anti-FIP200 (1:1000, Sigma, Cat#SAB4200135); mouse-anti-ATG16L1 (1:1000, MBL, Cat#PM040); rabbit-anti-IFT20 (1:1500, Proteintech, Cat#13615). Secondary HRP conjugate anti‐rabbit IgG (GE Healthcare) and HRP conjugate anti‐mouse IgG (Bio‐Rad).

Cells in microslides were washed twice with ice-cold PBS and lysed on ice with 150 μl of 1X Laemmli buffer (60 mM Tris-HCl pH=6.8, 2% SDS, 10% Glycerol, bromophenol blue, supplemented with 100 mM DTT) for 30 min. Samples were boiled for 10 min at 95°C, separated by SDS/PAGE and then transferred onto Nitrocellulose membranes. Western blot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore). The following antibodies were used in immunoblotting: rabbit-anti LC3 (Sigma, Cat#L7543); rabbit-anti-FLCN (Cell signaling, Cat#3697); rabbit-anti-FNIP1 (Abcam, Cat#ab134969); rabbit-anti-AMPK (Cell signaling, Cat#2532S); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); mouse-anti-actin (Millipore, Cat#1501); rabbit-anti-ATG16L1 (MBL, Cat#PM040); rabbit-anti-IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti β-catenin (Cell signaling, Cat#8480); rabbit-anti-LKB1 (Cell signaling, Cat#3050); rabbit-anti-S6 ribosomal protein (Cell signaling, Cat#2217); rabbit-anti-p-S6 ribosomal protein (S240/244) (Cell signaling, Cat#2215); rabbit-anti-Tuberin/TSC2 (Cell signaling, Cat#4308); rabbit-anti-p-Tuberin/TSC2 (T1462) (Cell signaling, Cat#3617). Secondary HRP conjugate anti-rabbit IgG (GE Healthcare) and HRP conjugate anti-mouse IgG (Bio-Rad).

Cells were fixed either with 4% paraformaldehyde (PFA) for 20 min or with cold methanol for 5 min at -20°C for proper axoneme proteins detection [22 (link)]. They were then washed and incubated for 30 min in blocking buffer (10% FCS in PBS) followed by incubation with primary antibodies diluted in blocking buffer supplemented with 0.05% saponin for 1 h at room temperature or overnight at 4°C. Cells were washed 3 times, and then incubated for 1 h with fluorescent Alexa-Fluor secondary antibodies. After washing, 150 μl of DAPI-Fluoromount were added into the Luer chamber (Southern Biotech). Images were acquired with a Zeiss Apotome.2 fluorescence microscope equipped with a 63x oil immersion fluorescence objective. Number of ciliated cells and length of cilia were quantified using Zen Software (Zeiss) or Imaris Software (Bitplane). The following antibodies were used for immunofluo-rescence: mouse-anti-LC3B (MBL, Cat# M152-3); rabbit-anti-FLCN (Cell signaling, Cat#3697); mouse-anti-ARL13B (C-5) (Santa Cruz, Cat#515784); rabbit-anti-ATG16 (MBL, Cat#PM040); rabbit-anti IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); Phalloidin (Cat# A34055); Alexa Fluor-conjugated secondary anti-bodies (donkey anti-mouse IgG and donkey anti-Rabbit IgG, Life Technologies).